Phytopathology • 2017 • 107:804-815 • http://dx.doi.org/10.1094/PHYTO-02-17-0047-RVW Rathayibacter toxicus,OtherRathayibacter Species Inducing Bacterial Head Blight of Grasses, and the Potential for Livestock Poisonings Timothy D. Murray, Brenda K. Schroeder, William L. Schneider, Douglas G. Luster, Aaron Sechler, Elizabeth E. Rogers, and Sergei A. Subbotin First author: Department of Plant Pathology, Washington State University, Pullman, WA 99164; second author: Entomology, Plant Pathology and Nematology, University of Idaho, Moscow, ID 83844; third, fourth, fifth, and sixth authors: U.S. Department of Agriculture, Agricultural Research Service, Foreign Disease-Weed Science Research Unit, Ft. Detrick, MD 21702; and seventh author: California Department of Food and Agriculture, 3294, Meadowview Road, Sacramento, CA 95832-1448. Accepted for publication 8 April 2017. ABSTRACT Rathayibacter toxicus, a Select Agent in the United States, is one of six recognized species in the genus Rathayibacter and the best known due to its association with annual ryegrass toxicity, which occurs only in parts of Australia. The Rathayibacter species are unusual among phytopathogenic bacteria in that they are transmitted by anguinid seed gall nematodes and produce extracellular polysaccharides in infected plants resulting in bacteriosis diseases with common names such as yellow slime and bacterial head blight. R. toxicus is distinguished from the other species by producing corynetoxins in infected plants; toxin production is associated with infection by a bacteriophage. These toxins cause grazing animals feeding on infected plants to develop convulsions and abnormal gate, which is referred to as “staggers,” and often results in death of affected animals. R. toxicus is the only recognized Rathayibacter species to produce toxin, although reports of livestock deaths in the United States suggest a closely related toxigenic species may be present. A closely related but undescribed species, Rathayibacter sp. EV, originally isolated from Ehrharta villosa var. villosa in South Africa, is suspected of producing toxin. Many of the diseases caused by Rathayibacter species occur in arid areas and the extracellular polysaccharide they produce is believed to aid in their survival between crops. For example, R. “agropyri” was isolated from infected plant material after being stored for 50 years in a herbarium. Similarly, the anguinid vectors associated with these bacteria form seed galls in infected plants and are capable of surviving for very long periods of time under dry conditions. The addition of R. toxicus to the list of Select Agents has raised concern over its potential introduction and a realization that current diagnostic methods are inadequate to distinguish among Rathayibacter species. In addition, little is known about the Rathayibacter species and their seed gall nematode vectors present in the United States. Additional keywords: bacteriology, ecology and epidemiology, etiology, nematology. Rathayibacter toxicus (Riley and Ophel 1992) Sasaki et al. 1998 are quarantine pests, to nearby host plants where it causes a was listed as a Plant Pathogen Select Agent under 7 CFR 331 by the bacteriosis disease of leaves and floral structures of several differ- U.S. Department of Agriculture (USDA) Animal and Plant Health ent grass species, but primarily annual ryegrass (Lolium rigidum Inspection Service in 2008 (Murray et al. 2015). Relatively few Gaud.). During colonization, R. toxicus produces corynetoxins that plant pathologists would have known of this bacterial plant can result in fatal poisoning of animals that graze on diseased plants, pathogen prior to its listing as a Select Agent, and fewer still would a syndrome now referred to as annual ryegrass toxicity (ARGT). To know that there are five other described species of Rathayibacter further complicate the relationship, Bird et al. (1980) and Stynes that cause similar diseases and have similar life cycles in other grass and Bird (1983) postulated the involvement of a bacteriophage in hosts. What is it about this Gram-positive bacterium that makes it toxin production. In Australia, this cross-domain pathogen has such a threat to be considered a Select Agent? R. toxicus is caused plant disease on over 10 million hectares with losses due to transmitted by seed gall nematodes (Anguina sp.), some of which ARGT in 2010 estimated at $37 million USD (Carslake 2006; Kessell 2010). The potential for damage to the U.S. livestock industry resulted in listing of R. toxicus as a Select Agent in 2008 Corresponding author: T. Murray; E-mail address: [email protected] and relisting in 2012. The National Plant Disease Recovery System (NPDRS) subsequently developed a recovery plan for this pathogen Rathayibacter This article is in the public domain and not copyrightable. It may be freely (Murray et al. 2015). None of the other five described reprinted with customary crediting of the source. The American Phytopathological species have been shown to produce toxins in planta, although Society, 2017. reports exist of livestock poisonings in the United States and 804 PHYTOPATHOLOGY elsewhere after animals grazed on pastures where seed gall nema- for sheep feeding trials. Postmortem examinations also were todes were present (Cunningham and Hartley 1959; Galey et al. conducted on animals that died of staggers from farms and feeding 1997; Galloway 1961; Haag 1945; Jensen 1961; Kurochkina and trials. They found that all pastures contained Wimmera ryegrass Chizhov 1980; Shaw and Muth 1949). (L. rigidum) and had generally high nematode infestations. One This review will examine the literature relevant to the history and such pasture with 21% infected seed had the greatest sheep biology of Rathayibacter broadly because they represent a fasci- mortality. Experimentally, sheep that grazed on one of the infested nating group of cross-domain pathogens that frequently occur in pastures developed symptoms typical of staggers and died, as arid areas (Fig. 1A). As such, both the pathogens and their vectors did guinea pigs fed bacteria-infected seed. All had postmortem have developed specialized mechanisms for anhydrobiotic survival, symptoms including uncoordinated movement and tetanic spasms namely production of extracellular polysaccharides (EPS) (Fig. 1B that were consistent with those of animals that died of staggers and C) and the formation of galls (Fig. 2A and B), in many of these described previously. McIntosh et al. (1967) could not conclusively diseases. Readers primarily interested in R. toxicus are directed demonstrate whether the toxicity was due to the nematode or the to the reviews by McKay and Ophel (1993), Riley et al. (2014), and bacterium, but concluded that both were associated with ryegrass the NPDRS Recovery Plan (Murray et al. 2015), which discuss staggers. Lanigan et al. (1976) followed-up this research with the relevant literature and provide recommendations for control and animal feeding studies and concluded that the bacterial galls and not surveillance. the nematode galls were the source of toxicity. Gwynn and Hadlow (1971) expanded the reports of toxicity to ANNUAL RYEGRASS TOXICITY sheep grazing on Wimmera ryegrass to include pastures in Western Australia. They noted that the disease had occurred in four of the In 1956, Fisher (1977) received samples of annual ryegrass six previous years in one particular pasture. Berry and Wise (1975) (L. rigidum) from veterinary officers with the South Australia coined the term Wimmera rye grass toxicity, or WRGT, for the Department of Agriculture that were taken from a pasture where syndrome in animals associated with grazing pastures infested with sheep had died after feeding on the grass. Fisher found seed gall seed gall nematodes and bacteria and noted that there were 58 nematodes present in the sample and, while visiting the field 2 years outbreaks on 26 farms with over 3,300 dead sheep and 46 cattle. Up later, observed symptoms of bacterial head blight, which he to 60% of the ryegrass sampled from the 26 farms was infected with attributed to a Corynebacterium species. Nematode seed galls both Anguina sp. and Corynebacterium sp. (now Rathayibacter), picked from the sample and fed to laboratory mice resulted in the neither of which were identified to species. death of one with symptoms similar to those of the dead sheep. By Bryden et al. (1991) described the occurrence of a syndrome 1967, increases in the numbers of sheep dying annually and affected known as flood plain staggers (FPS) in cattle and sheep in New properties in South Australia were great enough that funds were South Wales, Australia. Samples of blown grass (Agrostis avenacea obtained to begin new research on the problem at the Waite C.C. Gmelin) were collected and used in feeding studies that Agricultural Institute (Fisher 1977). resulted in animals developing symptoms similar to ARGT. Like McIntosh et al. (1967) were the first to conclude that a toxin was ARGT, a nematode and Clavibacter sp. (now Rathayibacter) were present where sheep were dying after feeding on infested pastures. found in seed heads, and a corynetoxin was extracted from the They surveyed nine farms where staggers occurred and sampled bacterial galls (Cockrum and Edgar 1985). Davis et al. (1995) later grass from four of them. Samples were identified to plant species, reported that 1,722 cattle, 2,466 sheep, and 11 horses on 31 different evaluated for presence of Anguina agrostis (Steinbuch 1799) properties died as a result of FPS